Method for designing a drug regime for hiv-infected patients

ABSTRACT

The instant disclosure describes a novel genotype and phenotype assay to elucidate and/or evaluate new potential HIV integrase inhibitors, but also currently approved and experimental compounds that target protease, reverse transcriptase, and RNaseH. This assay allows studying linked mutations and mutational patterns that occur under HAART and experimental therapies.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/617,855, filed onFeb. 9, 2015, which is a divisional application of U.S. application Ser.No. 13/668,906, filed Nov. 5, 2012 (now abandoned), which is adivisional application of U.S. application Ser. No. 12/524,120, filedJul. 22, 2009 (now U.S. Pat. No. 8,338,101, issued Dec. 25, 2012), whichis the national stage of PCT Application No. PCT/EP2008/050778 filedJan. 23, 2008, which claims priority from European Patent ApplicationNo. 07102423.6, filed Feb. 15, 2007, and European Patent Application No.07101037.5, filed Jan. 23, 2007, the entire disclosures of each of whichare hereby incorporated by reference.

BACKGROUND

Millions and millions of people have been infected with the humanimmunodeficiency virus (“HIV”), the causative agent of acquired immunedeficiency syndrome (“AIDS”), since the early 1980s. HIV/AIDS is now theleading cause of death in sub-Saharan Africa, and is the fourth biggestkiller worldwide. At the end of 2001, an estimated 40 million peoplewere living with HIV globally.

Currently, five classes of antiretroviral drugs are used to treatinfection by Human Immunodeficiency Virus (HIV), i.e. proteaseinhibitors (PIs), two classes of reverse transcriptase inhibitors(nucleoside reverse transcriptase inhibitors abbreviated as N RTI andnon-nucleoside reverse transcriptase inhibitors abbreviated as NN-RTI),entry inhibitors (fusion inhibitors (FIs) and co-receptor antagonists),and intergrase inhibitors (INIs). Integrase inhibitors are a promisingnew class of antiretrovirals interfering with HIV replication byblocking the ability of the virus to integrate into the genetic materialof human cells.

Modern anti-HIV drugs target different stages of the HIV life cycle anda variety of enzymes essential for HIV's replication and/or survival.Amongst the drugs that have so far been approved for AIDS therapy arenucleoside reverse transcriptase inhibitors (“NRTIs”) such as AZT, ddl,ddC, d4T, 3TC, and abacavir; nucleotide reverse transcriptase inhibitorssuch as tenofovir; non-nucleoside reverse transcriptase inhibitors(“NNRTIs”) such as nevirapine, efavirenz, and delavirdine; proteaseinhibitors (“PIs”) such as darunavir, saquinavir, ritonavir, indinavir,nelfinavir, amprenavir, lopinavir and atazanavir; fusion inhibitors,such as enfuvirtide, co-receptor antagonists such as maraviroc andintegrase inhibitors such as raltegravir.

Nonetheless, in the vast majority of subjects none of the antiviraldrugs currently approved, either alone or in combination, proveseffective either to prevent eventual progression of chronic HIVinfection to AIDS or to treat acute AIDS. This phenomenon is due, inpart, to the high mutation rate of HIV and the rapid emergence of mutantHIV that are resistant to antiviral therapeutics upon administration ofsuch drugs to infected individuals.

The integrase protein thus represents an interesting target for HIVinhibitor research. HIV integrase is required for integration of theviral genome into the genome of the host cell, a step in the replicativecycle of the virus. HIV integrase is a protein of about 32 KDa encodedby the pol gene, and is produced in vivo by protease cleavage of thegag-pol precursor protein during the production of viral particles. Theintegration process takes place following reverse transcription of theviral RNA. First, the viral integrase binds to the viral DNA and removestwo nucleotides from the 3′ end of the viral long-terminal repeat (LTR)sequences on each strand. This step is called 3′ end processing andoccurs in the cytoplasm within a nucleoprotein complex termed thepre-integration complex (PIC). Second, in a process called strandtransfer, the two strands of the cellular DNA into which the viral DNAwill be inserted, the target DNA, is cleaved in a staggered fashion. The3′ ends of the viral DNA are ligated to the 5′ ends of the cleavedtarget DNA. Finally, host enzymes probably repair remaining gaps.

With the increasing number of available anti-HIV compounds as mentionedabove, the number of potential treatment protocols for HIV infectedpatients will continue to increase. Many of the currently availablecompounds are administered as part of a combination therapy. The highcomplexity of treatment options coupled with the ability of the virus todevelop resistance to HIV inhibitors requires the frequent assessment oftreatment strategies. The ability to accurately monitor the replicativecapacity of virus in patients with a drug regimen and to use that datato modify the doses or combinations of inhibitors allows physicians toeffectively reduce the formation of drug resistant virus and provide anoptimal, tailored treatment for each patient.

Accordingly, as new drugs targeting new HIV polypeptides becomeavailable, phenotypic and genotypic assays for determining resistance orsusceptibility of HIV infecting a patient to such new anti-HIV drugs arehighly needed.

While phenotyping and genotyping assays have been developed and marketedfor reverse transcriptase and protease genes, protocols and assays forevaluation of drug resistance against the integrase gene have not beensuccessfully developed.

For instance, the amplicon used in the marketed Antivirogram® containsthe gag cleavage sites (p1/p7 and p1/p6), PR (codon 1-99) and RT (codon1-400) coding sequences respectively, leaving the rest of the relevantHIV reverse transcriptase gene and more importantly the HIV integrasegene undetected.

SUMMARY

The instant disclosure describes a novel genotype and phenotype assay toelucidate and/or evaluate new HIV integrase inhibitors, but alsocurrently approved and experimental compounds that target maturation,protease, reverse transcriptase, and RNaseH. This assay allows studyinglinked mutations and mutational patterns that occur under HAART andexperimental therapies. The selection of the primers used for thepreparation of the appropriate amplicon is, due to the mutations andmutational patterns present in the HIV sequence, of the utmostimportance to further develop a reliable and sensitive genotype andphenotype assay.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G show a schematic representation for creating the GAG-POLvector backbone based on the HXB2D_eGFP HIV-1 vector. FIGS. 1A-1D, 1F,and 1G illustrate certain digestion/ligation vector manipulations, whileFIG. 1E depicts PCR amplification of Fragment A.

FIG. 2 provides a more detailed schematic representation of the inversePCR described for creating the GAG-POL vector backbone based on theHXB2D_eGFP HIV-1 vector.

FIG. 3 depicts the primer binding sites for the inverse PCR describedfor creating the GAG-POL vector backbone based on the HXB2D_eGFP HIV-1vector.

FIGS. 4A-4G show a schematic representation for creating the RT-INTvector backbone based on the HXB2D_eGFP HIV-1 vector. FIGS. 4A-4D, 4F,and 4G illustrate certain digestion/ligation vector manipulations, whileFIG. 4E depicts PCR amplification of Fragment X.

FIG. 5 provides a more detailed schematic representation of the inversePCR described for creating the RT-INT vector backbone based on theHXB2D_eGFP HIV-1 vector.

FIG. 6 depicts the primer binding sites for the inverse PCR describedfor creating the RT-INT vector backbone based on the HXB2D_eGFP HIV-1vector.

FIGS. 7A-7G show a schematic representation for creating the GAG-PRvector backbone based on the HXB2D_eGFP HIV-1 vector. FIGS. 7A-7D, 7F,and 7G illustrate certain digestion/ligation vector manipulations, whileFIG. 7E depicts PCR amplification of Fragment A.

FIG. 8 provides a more detailed schematic representation of the inversePCR described for creating the GAG-PR vector backbone based on theHXB2D_eGFP HIV-1 vector.

FIG. 9 depicts the primer binding sites for the inverse PCR describedfor creating the GAG-PR vector backbone based on the HXB2D_eGFP HIV-1vector.

FIG. 10 is a flow chart summarizing an experimental process fordetermining the phenotype of viruses produced using a GAG-POL, GAG-PR orRT-INT vector.

FIGS. 11A-11R show the dose-response curves 1 GAG-POL RVS for all drugstested.

FIGS. 12A-12G show a schematic representation for creating thedelta[POL] vector backbone based on the HXB2D_eGFP HIV-1 vector. FIGS.12A-12D, 12F, and 12G illustrate certain digestion/ligation vectormanipulations, while FIG. 12E depicts PCR amplification of Fragment A.

FIG. 13 depicts the primer binding sites for the inverse PCR describedfor creating the delta[POL] vector backbone based on the HXB2D_eGFPHIV-1 vector.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In contrast to the amplicon mentioned above as used in the Antivirogram,the amplicon described in the instant invention and referred to as 5′LTR-Vif fragment contains the complete gag and complete pol (PR-RT-INT)coding region (4588 bp in HXB2D, GenBank accession number K03455).

Gag is the Group-specific Antigen protein, encoding the structuralcapsid proteins. The proteins are produced as a GAG precursorpolyprotein, which is processed by the viral protease.

Other amplicons used in the current invention are the amplicon spanningthe Gag cleavage sites p1/p7 and p1/p6, PR, RT, RNaseH and INT (3202bp), referred to as Pol fragment, the amplicon containing the Gag and PRcoding sequence (1980 bp), referred to as Gag-PR fragment, and theamplicon containing the complete RT, RNaseH and INT coding sequence(2898 bp), named RT-INT fragment.

The current disclosure describes an in vitro method for designing a drugregimen for an HIV-infected patient by determining the phenotypicsusceptibility of HIV to at least one drug, comprising:

i) using at least one sample comprising HIV RNA from a patient, whereinthe sample comprises the complete HIV gag-pol coding region;

ii) reverse-transcribing and amplifying the HIV RNA with primersspecific for the complete HIV gag-pol coding region to obtain at leastone amplicon comprising the complete HIV gag-pol coding region, whereinat least one primer is selected from SEQ ID NO: 1-4;

iii) generating a plasmid comprising a reference HIV sequence with adeletion of the complete HIV gag-pol coding region;

iv) preparing at least one recombinant virus by recombination orligation between at least one amplicon obtained in step ii) and theplasmid comprising the reference HIV sequence with a deletion of thecomplete HIV gag-pol coding region obtained in step iii), and

v) monitoring the at least one recombinant virus in the presence of theat least one drug to determine the phenotypic susceptibility of HIV toat least one drug,

wherein said susceptibility is determined by the cytopathogenicity ofsaid recombinant virus to cells or by determining the replicativecapacity of said recombinant virus in the presence of at least one drug.

The instant disclosure describes an in vitro method for designing a drugregimen for an HIV-infected patient by determining the phenotypicsusceptibility of HIV to at least one drug, comprising:

i) using at least one sample comprising HIV RNA from a patient, whereinthe sample comprises the region spanning the HIV gag-protease codingsequence;

ii) reverse-transcribing and amplifying the HIV RNA with primersspecific for the region spanning the HIV gag-protease coding sequence toobtain at least one amplicon comprising the region spanning the HIVgag-protease coding sequence, wherein at least one primer is selectedfrom SEQ ID NO: 1 and SEQ ID NO: 8-10;

iii) generating a plasmid comprising a reference HIV sequence with adeletion of the region spanning the HIV gag-protease coding sequence;

iv) preparing at least one recombinant virus by recombination orligation between at least one amplicon obtained in step ii) and theplasmid comprising the reference HIV sequence with a deletion of theregion spanning the HIV gag-protease coding sequence obtained in stepiii), and

v) monitoring the at least one recombinant virus in the presence of theat least one drug to determine the phenotypic susceptibility of HIV toat least one drug,

wherein said susceptibility is determined by the cytopathogenicity ofsaid recombinant virus to cells or by determining the replicativecapacity of said recombinant virus in the presence of at least one drug.

Furthermore the present disclosure also comprises an in vitro method fordesigning a drug regimen for an HIV-infected patient by determining thephenotypic susceptibility of HIV to at least one drug, comprising:

i) using at least one sample comprising HIV RNA from a patient, whereinthe sample comprises the complete HIV reverse transcriptase-integrasecoding sequence;

ii) reverse-transcribing and amplifying the HIV RNA with primersspecific for the complete HIV reverse transcriptase-integrase codingsequence to obtain at least one amplicon comprising the complete HIVreverse transcriptase-integrase coding sequence, wherein at least oneprimer is selected from SEQ ID NO: 4-7;

iii) generating a plasmid comprising a reference HIV sequence with adeletion of the complete HIV reverse transcriptase-integrase codingsequence;

iv) preparing at least one recombinant virus by recombination orligation between at least one amplicon obtained in step ii) and theplasmid comprising the reference HIV sequence with a deletion of thecomplete HIV reverse transcriptase-integrase coding sequence obtained instep iii), and

v) monitoring the at least one recombinant virus in the presence of theat least one drug to determine the phenotypic susceptibility of HIV toat least one drug,

wherein said susceptibility is determined by the cytopathogenicity ofsaid recombinant virus to cells or by determining the replicativecapacity of said recombinant virus in the presence of at least one drug.

The current invention also applies to an in vitro method for designing adrug regimen for an HIV-infected patient by determining the phenotypicsusceptibility of HIV to at least one drug, comprising:

i) using at least one sample comprising HIV DNA, wherein the samplecomprises the complete HIV gag-pol coding region;

ii) amplifying the HIV DNA with primers specific for the complete HIVgag-pol coding region to obtain at least one amplicon comprising thecomplete HIV gag-pol coding region, wherein at least one primer isselected from SEQ ID NO: 1-4;

iii) generating a plasmid comprising a reference HIV sequence with adeletion of the complete HIV gag-pol coding region;

iv) preparing at least one recombinant virus by recombination orligation between at least one amplicon obtained in step ii) and theplasmid comprising the reference HIV sequence with a deletion of thecomplete HIV gag-pol coding region obtained in step iii), and

v) monitoring the at least one recombinant virus in the presence of theat least one drug to determine the phenotypic susceptibility of HIV toat least one drug,

wherein said susceptibility is determined by the cytopathogenicity ofsaid recombinant virus to cells or by determining the replicativecapacity of said recombinant virus in the presence of at least one drug.

In addition the disclosure describes an in vitro method for designing adrug regimen for an HIV-infected patient by determining the phenotypicsusceptibility of HIV to at least one drug, comprising:

i) using at least one sample comprising HIV DNA, wherein the samplecomprises the region spanning the HIV gag-protease coding sequence;

ii) amplifying the HIV DNA with primers specific for the region spanningthe HIV gag-protease coding sequence to obtain at least one ampliconcomprising the region spanning the HIV gag-protease coding sequence,wherein at least one primer is selected from SEQ ID NO: 1 and SEQ ID NO:8-10;

iii) generating a plasmid comprising a reference HIV sequence with adeletion of the region spanning the HIV gag-protease coding sequence;

iv) preparing at least one recombinant virus by recombination orligation between at least one amplicon obtained in step ii) and theplasmid comprising the reference HIV sequence with a deletion of theregion spanning the HIV gag-protease coding sequence obtained in stepiii), and

v) monitoring the at least one recombinant virus in the presence of theat least one drug to determine the phenotypic susceptibility of HIV toat least one drug,

wherein said susceptibility is determined by the cytopathogenicity ofsaid recombinant virus to cells or by determining the replicativecapacity of said recombinant virus in the presence of at least one drug.

The disclosure also comprises an in vitro method for designing a drugregimen for an HIV-infected patient by determining the phenotypicsusceptibility of HIV to at least one drug, comprising:

i) using at least one sample comprising HIV DNA, wherein the samplecomprises the complete HIV reverse transcriptase-integrase codingsequence;

ii) amplifying the HIV DNA with primers specific for the complete HIVreverse transcriptase-integrase coding sequence to obtain at least oneamplicon comprising the complete HIV reverse transcriptase-integrasecoding sequence, wherein at least one primer is selected from SEQ ID NO:4-7;

iii) generating a plasmid comprising a reference HIV sequence with adeletion of the complete HIV reverse transcriptase-integrase codingsequence;

iv) preparing at least one recombinant virus by recombination orligation between at least one amplicon obtained in step ii) and theplasmid comprising the reference HIV sequence with a deletion of thecomplete HIV reverse transcriptase-integrase coding sequence obtained instep iii), and

v) monitoring the at least one recombinant virus in the presence of theat least one drug to determine the phenotypic susceptibility of HIV toat least one drug,

wherein said susceptibility is determined by the cytopathogenicity ofsaid recombinant virus to cells or by determining the replicativecapacity of said recombinant virus in the presence of at least one drug.

A further embodiment of the invention is an in vitro method fordetermining the phenotypic susceptibility of HIV to at least one drug,comprising:

i) using at least one sample comprising HIV RNA from a patient, whereinthe sample comprises the complete HIV gag-pol coding region;

ii) reverse-transcribing and amplifying said HIV RNA with primersspecific for the complete HIV gag-pol coding region to obtain anamplicon comprising the complete HIV gag-pol coding region, wherein atleast one primer is selected from SEQ ID NO: 1-4;

iii) determining the nucleotide sequence of the amplicon or a portionthereof as obtained in step ii), and

iv) comparing the nucleotide sequence of the amplicon with the sequenceof sequences whose phenotypic susceptibility is known to estimate thephenotypic susceptibility of HIV to at least one drug,

wherein said susceptibility is determined by the cytopathogenicity ofsaid recombinant virus to cells or by determining the replicativecapacity of said recombinant virus in the presence of at least one drug.

Part of the invention is also wherein the embodiment is an in vitromethod for determining the phenotypic susceptibility of HIV to at leastone drug, comprising:

i) using at least one sample comprising HIV RNA from a patient, whereinthe sample comprises the region spanning the HIV gag-protease codingsequence;

ii) reverse-transcribing and amplifying said HIV RNA with primersspecific for the region spanning the HIV gag-protease coding sequence toobtain an amplicon comprising the region spanning the HIV gag-proteasecoding region, wherein at least one primer is selected from SEQ ID NO: 1and SEQ ID NO: 8-10;

iii) determining the nucleotide sequence of the amplicon or a portionthereof as obtained in step ii), and

iv) comparing the nucleotide sequence of the amplicon with the sequenceof sequences whose phenotypic susceptibility is known to estimate thephenotypic susceptibility of HIV to at least one drug,

wherein said susceptibility is determined by the cytopathogenicity ofsaid recombinant virus to cells or by determining the replicativecapacity of said recombinant virus in the presence of at least one drug.

In addition the invention relates to an in vitro method for determiningthe phenotypic susceptibility of HIV to at least one drug, comprising:

i) using at least one sample comprising HIV RNA from a patient, whereinthe sample comprises the complete HIV reverse transcriptase-integrasecoding sequence;

ii) reverse-transcribing and amplifying said HIV RNA with primersspecific for the complete HIV reverse transcriptase-integrase codingsequence to obtain an amplicon comprising the complete HIV reversetranscriptase-integrase coding region, wherein at least one primer isselected from SEQ ID NO: 4-7;

iii) determining the nucleotide sequence of the amplicon or a portionthereof as obtained in step ii), and

iv) comparing the nucleotide sequence of the amplicon with the sequenceof sequences whose phenotypic susceptibility is known to estimate thephenotypic susceptibility of HIV to at least one drug,

wherein said susceptibility is determined by the cytopathogenicity ofsaid recombinant virus to cells or by determining the replicativecapacity of said recombinant virus in the presence of at least one drug.

To the invention also belongs an in vitro method for determining thephenotypic susceptibility of HIV to at least one drug, comprising:

i) using at least one sample comprising HIV DNA wherein the samplecomprises the complete HIV gag-pol coding region;

ii) amplifying said HIV DNA with primers specific for the complete HIVgag-pol coding region to obtain an amplicon comprising the complete HIVgag-pol coding region, wherein at least one primer is selected from SEQID NO: 1-4;

iii) determining the nucleotide sequence of the amplicon or a portionthereof as obtained in step ii), and

iv) comparing the nucleotide sequence of the amplicon with the sequenceof sequences whose phenotypic susceptibility is known to estimate thephenotypic susceptibility of HIV to at least one drug,

wherein said susceptibility is determined by the cytopathogenicity ofsaid recombinant virus to cells or by determining the replicativecapacity of said recombinant virus in the presence of at least one drug.

The above embodiment of the invention can be extended to an in vitromethod for determining the phenotypic susceptibility of HIV to at leastone drug using at least one sample comprising HIV DNA wherein the samplecomprises the region spanning the HIV gag-protease coding sequence usingthe appropriate primers SEQ ID NO: 1 and SEQ ID NO: 8-10 or wherein thesample comprises the complete HIV reverse transcriptase-integrase codingregion using the appropriate primers selected from SEQ ID NO 4-7respectively.

Part of the invention is also an in vitro method for designing a drugregimen for an HIV-infected patient by determining the phenotypicsusceptibility of HIV to at least one drug, comprising:

i) using at least one sample comprising HIV RNA from a patient, whereinthe sample comprises the complete HIV pol coding region;

ii) reverse-transcribing and amplifying the HIV RNA with primersspecific for the complete HIV pol coding region to obtain at least oneamplicon comprising the complete HIV pol coding region, wherein at leastone primer is selected from SEQ ID NO's: 2, 4, 53 and 54;

iii) generating a plasmid comprising a reference HIV sequence with adeletion of the complete HIV pol coding region;

iv) preparing at least one recombinant virus by recombination orligation between at least one amplicon obtained in step ii) and theplasmid comprising the reference HIV sequence with a deletion of thecomplete HIV pol coding region obtained in step iii), and

v) monitoring at least one recombinant virus in the presence of at leastone drug to determine the phenotypic susceptibility of HIV to at leastone drug,

wherein said susceptibility is determined by the cytopathogenicity ofsaid recombinant virus to cells or by determining the replicativecapacity of said recombinant virus in the presence of at least one drug.

In addition also to the invention belongs an in vitro method fordesigning a drug regime for an HIV-infected patient by determining thephenotypic susceptibility of HIV to at least one drug, comprising:

i) using at least one sample comprising HIV DNA, wherein the samplecomprises the complete HIV pol coding region;

ii) amplifying the HIV DNA with primers specific for the complete HIVpol coding region to obtain at least one amplicon comprising thecomplete HIV pol coding region, wherein at least one primer is selectedfrom SEQ ID NO: 2, 4, 53 and 54;

iii) generating a plasmid comprising a reference HIV sequence with adeletion of the complete HIV pol coding region;

iv) preparing at least one recombinant virus by recombination orligation between at least one amplicon obtained in step ii) and theplasmid comprising the reference HIV sequence with a deletion of thecomplete HIV pol coding region obtained in step iii), and

v) monitoring at least one recombinant virus in the presence of at leastone drug to determine the phenotypic susceptibility of HIV to at leastone drug,

wherein said susceptibility is determined by the cytopathogenicity ofsaid recombinant virus to cells or by determining the replicativecapacity of said recombinant virus in the presence of at least one drug

The disclosure further describes a method of constructing a genotypicand phenotypic database of HIV sequences, comprising:

i) using samples of HIV RNA from a patient comprising the complete HIVgag-pol coding region;

ii) reverse-transcribing and amplifying said HIV RNA with primersspecific for the complete HIV gag-pol coding region to obtain anamplicon comprising the complete HIV gag-pol coding region, wherein atleast one primer is selected from SEQ ID NO: 1-4;

iii) determining the nucleotide sequence of the amplicon or portionsthereof as obtained in step ii);

iv) generating a plasmid comprising a reference HIV sequence with adeletion of the complete HIV gag-pol coding region;

v) preparing recombinant virus by recombination or ligation between theamplicon obtained in step ii) and the plasmid comprising the referenceHIV sequence with a deletion of the complete HIV gag-pol coding regionobtained in step iv);

vi) determining the relative replicative capacity of the recombinantvirus in the presence of anti-HIV drugs compared to an HIV with areference complete HIV gag-pol coding region.

The disclosure also comprises an in vitro method of constructing agenotypic and phenotypic database of HIV sequences, comprising:

i) using samples of HIV DNA comprising the complete HIV gag-pol codingregion;

ii) amplifying said HIV DNA with primers specific for the complete HIVgag-pol coding region to obtain an amplicon comprising the complete HIVgag-pol coding region, wherein at least one primer is selected from SEQID NO: 1-4;

iii) determining the nucleotide sequence of the amplicon or portionsthereof as obtained in step ii);

iv) generating a plasmid comprising a reference HIV sequence with adeletion of the complete HIV gag-pol coding region;

v) preparing recombinant virus by recombination or ligation between theamplicon obtained in step ii) and the plasmid comprising the referenceHIV sequence with a deletion of the complete HIV gag-pol coding regionobtained in step iv);

vi) determining the relative replicative capacity of the recombinantvirus in the presence of anti-HIV drugs compared to an HIV virus with areference complete HIV gag-pol coding region.

The above embodiments of the invention of constructing a genotypic andphenotypic database of HIV sequences can be extended to using at leastone sample comprising either HIV RNA or DNA wherein the sample comprisesthe region spanning the HIV gag-protease coding sequence using theappropriate primers SEQ ID NO: 1 and SEQ ID NO: 8-10 or wherein thesample comprises the complete HIV reverse transcriptase-integrase codingregion using the appropriate primers selected from SEQ ID NO 4-7respectively.

Part of the invention is also a method of constructing a genotypic andphenotypic database of HIV sequences, comprising:

i) using samples of HIV RNA from a patient comprising the complete HIVpol coding region;

ii) reverse-transcribing and amplifying said HIV RNA with primersspecific for the complete HIV pol coding region to obtain an ampliconcomprising the complete HIV pol coding region, wherein at least oneprimer is selected from SEQ ID NO: 2, 4, 53 and 54;

iii) determining the nucleotide sequence of the amplicon or portionsthereof as obtained in step ii);

iv) generating a plasmid comprising a reference HIV sequence with adeletion of the complete HIV pol coding region;

v) preparing recombinant virus by recombination or ligation between theamplicon obtained in step ii) and the plasmid comprising the referenceHIV sequence with a deletion of the complete HIV pol coding regionobtained in step iv);

vi) determining the relative replicative capacity of the recombinantvirus in the presence of anti-HIV drugs compared to an HIV with areference complete HIV pol coding region.

In addition to the invention also belongs an in vitro method ofconstructing a genotypic and phenotypic database of HIV sequences,comprising:

i) using samples of HIV DNA comprising the complete HIV pol codingregion;

ii) amplifying said HIV DNA with primers specific for the complete HIVpol coding region to obtain an amplicon comprising the complete HIV polcoding region, wherein at least one primer is selected from SEQ ID NO:2, 4, 53 and 54;

iii) determining the nucleotide sequence of the amplicon or portionsthereof as obtained in step ii);

iv) generating a plasmid comprising a reference HIV sequence with adeletion of the complete HIV pol coding region;

v) preparing recombinant virus by recombination or ligation between theamplicon obtained in step ii) and the plasmid comprising the referenceHIV sequence with a deletion of the complete HIV pol coding regionobtained in step iv);

vi) determining the relative replicative capacity of the recombinantvirus in the presence of anti-HIV drugs compared to an HIV virus with areference complete HIV pol coding region.

The present invention also comprises the plasmids or sometimes calledvectors described in the experimental part and the use of these plasmidsor vectors in the methods described herein. The HIV sequenceincorporated in the plasmid or vector may be based on the K03455sequence. The complete HIV sequence may be incorporated or only partthereof. A suitable plasmid backbone may be selected from the groupincluding pUC, pSV or pGEM.

To prepare vectors containing recombinant HIV gag-pol coding sequences,the patient derived gag-pol RNA was reverse transcribed and amplified bythe polymerase chain reaction (PCR), then inserted into a vectorcontaining the wild type HIV genome sequence but lacking a completegag-pol coding region. Different primer combinations were initially usedto obtain the amplified DNA sequences from patient samples. The 5′ to 3′sequences and the primers identified as SEQ ID's NO: 1-10, morespecifically SEQ ID NO's: 1-4 were successfully used to reversetranscribe and PCR amplify gag-pol coding region are listed below inTable 7.

To prepare a vector containing recombinant HIV gag-protease codingsequence, the patient derived gag-protease RNA was reverse transcribedand amplified by the polymerase chain reaction (PCR), then inserted intoa vector containing the wild type HIV genome sequence but lackinggag-protease coding region. Different primer combinations were initiallyused to obtain the amplified DNA sequences from patient samples. The 5′to 3′ sequences and the primers identified as SEQ ID's NO: 1-10, morespecifically SEQ ID NO: 1 and SEQ ID NO's 8-10 were successfully used toreverse transcribe and PCR amplify gag-protease coding region are listedbelow in Table 7.

To prepare a vector containing recombinant HIV reversetranscriptase-integrase coding sequence, the patient derived reversetranscriptase-integrase RNA was reverse transcribed and amplified by thepolymerase chain reaction (PCR), then inserted into a vector containingthe wild type HIV genome sequence but lacking reversetranscriptase-integrase coding region. Different primer combinationswere initially used to obtain the amplified DNA sequences from patientsamples. The 5′ to 3′ sequences and the primers identified as SEQ ID'sNO: 1-10, more specifically SEQ ID NO: 4-7 were successfully used toreverse transcribe and PCR amplify reverse transcriptase-integrasecoding region are listed below in Table 7.

To prepare a vector containing recombinant HIV pol coding sequences, thepatient derived pol RNA was reverse transcribed and amplified by thepolymerase chain reaction (PCR), then inserted into a vector containingthe wild type HIV genome sequence but lacking a complete pol codingregion. Different primer combinations were initially used to obtain theamplified DNA sequences from patient samples. The 5′ to 3′ sequences andthe primers identified as SEQ ID's NO: 2, 4, 53 and 54 were successfullyused to reverse transcribe and PCR amplify pol coding region and arelisted below in Table 7.

Reverse transcription and amplification may be performed with a singleset of primers. Alternatively, more than one set of primers may be usedin a hemi-nested approach to reverse transcribe and amplify the geneticmaterial. Particularly, more than one set of primer is used in a nestedapproach. Following the generation of the recombinant construct, thechimeric virus may be grown and the viral titer determined (expressed as50% cell culture infectious dose, CCID50) before proceeding to thedetermination of the phenotypic susceptibility.

“Chimeric” means a construct comprising nucleic acid material fromdifferent origin such as for example a combination of wild type HIV witha laboratory HIV virus, a combination of wild type HIV sequence andpatient derived HIV sequence.

The indicator gene, encoding a signal indicative of replication of thevirus in the presence of a drug or indicative of the susceptibility ofthe virus in the presence of a drug may be present in the culturingcells such as MT-4 cells. In addition, said indicator gene may beincorporated in the chimeric construct introduced into the culturingcells or may be introduced separately. Suitable indicator genes encodefluorescent proteins, particularly green fluorescent protein (GFP) ormutants thereof such as eGFP (enhanced GFP).

Genetic material may be introduced into the cells using a variety oftechniques known in the art including, calcium phosphate precipitation,liposomes, viral infection, and electroporation. The monitoring may beperformed in high throughput.

A human immunodeficiency virus (HIV), as used herein refers to any HIVincluding laboratory HIV strains, wild type HIV strains, mutant HIVstrains and any biological sample comprising HIV such as a HIV clinicalisolate. HIV strains compatible with the present invention are thosestrains capable of infecting mammals, particularly humans such as HIV-1and HIV-2. A patient may have HIV in his body with different mutationsin the integrase (IN) gene. It is to be understood that a sample maycontain a variety of different HIV containing different mutationalprofiles in the IN gene. A sample may be obtained for example from anindividual, from cell cultures, or generated using recombinanttechnology, or cloning. Viral strains used for obtaining a plasmid arepreferably HIV wild-type sequences, such as LAI or HXB2D. LAI, alsoknown as IIIB, is a wild type HIV strain. One particular clone thereof,this means one sequence, is HXB2D. This sequence may be incorporatedinto a plasmid.

Instead of viral RNA, HIV DNA, e.g. proviral DNA, may be used for themethods described herein. In case RNA is used, reverse transcriptioninto DNA by a suitable reverse transcriptase is needed. The protocolsdescribing the analysis of RNA are also amenable for DNA analysis.However, if a protocol starts from DNA, the person skilled in the artwill know that no reverse transcription is needed. The primers designedto amplify the RNA strand, also anneal to, and amplify DNA. Reversetranscription and amplification may be performed with a single set ofprimers. Suitably a hemi-nested and more suitably a nested approach maybe used to reverse transcribe and amplify the genetic material.

Nucleic acid may be amplified by techniques such as polymerase chainreaction (PCR), nucleic acid sequence based amplification (NASBA),self-sustained sequence replication (3SR), transcription-basedamplification (TAS), ligation chain reaction (LCR). Preferably thepolymerase chain reaction is used.

Any type of patient sample may be used to obtain the integrase gene,such as serum or tissue. Viral RNA may be isolated using known methodssuch as described in Boom, R. et al. (J. Clin. Microbiol. 28(3): 495-503(1990)). Alternatively, a number of commercial methods such as theQIAAMP® viral RNA kit (Qiagen, Inc.) or EasyMag RNA extraction platform(Biomérieux, Boxtel, the Netherlands) may be used to obtain viral RNAfrom bodily fluids such as plasma, serum, or cell-free fluids. DNA maybe obtained by procedures known in the art (e.g. Maniatis, 1989) andcommercial procedures (e.g. Qiagen).

According to the instant invention, for instance, the complete HIV gagand complete pol (Protease-reverse transcriptase-integrase) codingregion (4588 bp) is used to prepare an amplicon.

“Amplicon” refers to the amplified, and where necessary, reversetranscribed complete gag-protease-reverse transcriptase-integrasesequence.

It should be understood that this complete gag-protease-reversetranscriptase-integrase sequence may be of diverse origin includingplasmids and patient material. Suitably, the amplicon is obtained frompatient material.

For the purpose of the present invention the amplicon is sometimesreferred to as “DNA construct”. A viral sequence may contain one ormultiple mutations versus the consensus reference sequence given byHXB2D, GenBank accession number K03455. Said sequence, K03455, ispresent in Genbank and available through the Internet. A single mutationor a combination of mutations may correlate to a change in drugefficacy. This correlation may be indicative of an altered i.e.decreased or increased susceptibility of the virus for a drug. Saidmutations may also influence the viral fitness.

A “drug” means any agent such as a chemotherapeutic, peptide, antibody,antisense, ribozyme and any combination thereof. Examples of drugsinclude protease inhibitors including darunavir, ritonavir, amprenavir,nelfinavir; reverse transcriptase inhibitors such as nevirapine,delavirdine, AZT, zidovudine, didanosine; integrase inhibitors; agentsinterfering with envelope (such as T-20).

Treatment or treatment regimen refers to the therapeutic management ofan individual by the administration of drugs. Different drug dosages,administration schemes, administration routes and combinations may beused to treat an individual.

An alteration in viral drug sensitivity is defined as a change insusceptibility of a viral strain to said drug. Susceptibilities aregenerally expressed as ratios of EC₅₀ or EC₉₀ values (the EC₅₀ or EC₉₀value being the drug concentration at which 50% or 90% respectively ofthe viral population is inhibited from replicating) of a viral strainunder investigation compared to the wild type strain. Hence, thesusceptibility of a viral strain towards a certain drug can be expressedas a fold change in susceptibility, wherein the fold change is derivedfrom the ratio of for instance the EC₅₀ values of a mutant viral straincompared to the wild type EC₅₀ values. In particular, the susceptibilityof a viral strain or population may also be expressed as resistance of aviral strain, wherein the result is indicated as a fold increase in EC₅₀as compared to wild type IC₅₀.

The IC₅₀ is the drug concentration at which 50% of the enzyme activityis inhibited.

The susceptibility of HIV to a drug is tested by either determining thecytopathogenicity of the recombinant virus to cells or by determiningthe replicative capacity of the recombinant virus in the presence of atleast one drug, relative to the replicative capacity of a wild type orreference HIV.

In the context of this invention, the cytopathogenic effect means theviability of the cells in culture in the presence of chimeric viruses.The cells may be chosen from T cells, monocytes, macrophages, dendriticcells, Langerhans cells, hematopoetic stem cells or precursor cells, MT4cells and PM-1 cells. The cytopathogenicity may, for example, befollowed microscopically, or replication might be monitored by thepresence of any reporter molecule including reporter genes. A reportergene is defined as a gene whose product has reporting capabilities.Suitable reporter molecules include tetrazolium salts, green fluorescentproteins, beta-galactosidase, chloramfenicol transferase, alkalinephophatase, and luciferase. Several methods of cytopathogenic testingincluding phenotypic testing are described in the literature comprisingthe recombinant virus assay (Kellam and Larder, Antimicrob. AgentsChemotherap. 1994, 38, 23-30, Hertogs et al. Antimicrob. AgentsChemotherap. 1998, 42, 269-276; Pauwels et al. J. Virol Methods 1988,20, 309-321)

The susceptibility of HIV to a drug may also be determined by thereplicative capacity of the recombinant virus in the presence of atleast one drug, relative to the replicative capacity of a reference orwild type HIV. Replicative capacity means the ability of the virus orchimeric construct to grow under culturing conditions. This is sometimesreferred to as viral fitness. The culturing conditions may containtriggers that influence the growth of the virus, examples of which aredrugs. The methods for determining the susceptibility may be useful fordesigning a treatment regimen for an HIV-infected patient. For example,a method may comprise determining the replicative capacity of a clinicalisolate of a patient and using said replicative capacity to determine anappropriate drug regime for the patient.

The phenotyping assays of the present invention can be performed at highthroughput using, for example, a microtiter plate containing a varietyof anti-HIV drugs. The present assays may be used to analyze theinfluence of changes at the HIV gag-pol gene to any type of drug usefulto treat HIV. Examples of anti-HIV drugs that can be tested in thisassay include, nucleoside and non-nucleoside reverse transcriptaseinhibitors, nucleotide reverse transcriptase inhibitors, proteaseinhibitors, maturation inhibitors, RNaseH inhibitors and integraseinhibitors, but those of skill in the art will appreciate that othertypes of antiviral compounds may also be tested. The results may bemonitored by several approaches including but not limited to morphologyscreening, microscopy, and optical methods, such as, for example,absorbance and fluorescence. An IC₅₀ value for each drug may be obtainedin these assays and used to determine viral replicative capacity in thepresence of the drug. Apart from IC₅₀ also e.g. IC₉₀ can be used. Thereplicative capacity of the viruses may be compared to that of awild-type HIV virus to determine a relative replicative capacity value.Data from phenotypic assays may further be used to predict the behaviourof a particular HIV isolate to a given drug based on its genotype.

The assays of the present invention may be used for therapeutic drugmonitoring. Said approach includes a combination of susceptibilitytesting, determination of drug level and assessment of a threshold. Saidthreshold may be derived from population based pharmacokinetic modelling(WO 02/23186). The threshold is a drug concentration needed to obtain abeneficial therapeutic effect in vivo. The in vivo drug level may bedetermined using techniques such as high performance liquidchromatography, liquid chromatography, mass spectroscopy or combinationsthereof. The susceptibility of the virus may be derived from phenotypingor interpretation of genotyping results i.e. virtual phenotyping (WO01/79540).

The assays of the present invention may be useful to discriminate aneffective drug from an ineffective drug by establishing cut-offs i.e.biological cut-offs (see e.g. WO 02/33402). A biological cut-off is drugspecific. These cut-offs are determined following phenotyping a largepopulation of individuals containing wild type viruses. The cut-off isderived from the distribution of the fold increase in resistance of thevirus for a particular drug.

The genotype of the patient-derived gag-pol coding region may bedetermined directly from the amplified DNA, i.e. the DNA construct byperforming DNA sequencing. Alternatively, the sequence may be obtainedafter sub-cloning into a suitable vector. A variety of commercialsequencing enzymes and equipment may be used in this process. Theefficiency may be increased by determining the sequence of the gag-polcoding region in several parallel reactions, each with a different setof primers. Such a process could be performed at high throughput on amultiple-well plate, for example. Commercially available detection andanalysis systems may be used to determine and store the sequenceinformation for later analysis.

The nucleotide sequence may be obtained using several approachesincluding sequencing nucleic acids. This sequencing may be performedusing techniques including gel based approaches, mass spectroscopy andhybridisation. However, as more resistance related mutations areidentified, the sequence at particular nucleic acids, codons or shortsequences may be obtained. If a particular resistance associatedmutation is known, the nucleotide sequence may be determined usinghybridisation assays (including Biochips, LipA-assay), massspectroscopy, allele specific PCR, or using probes or primersdiscriminating between mutant and wild-type sequence. A selected set ofsequencing primers includes SEQ ID No's: 11-44 and 55-58 respectively(Table 10). This particular selection has the advantage that it enablesthe sequencing of the complete HIV gag-pol coding sequence.Consequently, using this set of primers all possible mutations that mayoccur in the HIV gag or pol gene may be detected.

The patient gag-pol genotype provides an additional means to determinedrug susceptibility of a virus strain. Phenotyping is a lengthy processoften requiring 2 or more weeks to accomplish. Therefore, systems havebeen developed which enable the prediction of the phenotype based on thegenotypic results. The results of genotyping may be interpreted inconjunction with phenotyping and eventually be subjected to databaseinterrogation. A suitable system is virtual phenotyping (WO 01/79540).In the virtual phenotyping process the complete gag-pol genes may beused. Alternatively, portions of the genes may be used. Alsocombinations of mutations, preferentially mutations indicative of achange in drug susceptibility, may be used. A combination of mutationsis sometimes referred to as a hot-spot (see e.g. WO 01/79540). Briefly,in the process of virtual phenotyping, the genotype of a patient derivedgag-pol sequence may be correlated to the phenotypic response of saidpatient derived gag-pol sequence. If no phenotyping is performed, thesequence may be screened towards a collection of sequences present in adatabase. Identical sequences are retrieved and the database is furtherinterrogated to identify if a corresponding phenotype is known for anyof the retrieved sequences. In this latter case a virtual phenotype maybe determined. A report may be prepared including the IC₅₀ of the viralstrain for one or more therapies, the sequence of the strain underinvestigation, and the biological cut-offs.

According to the methods described herein a database may be constructedcomprising genotypic and phenotypic data of the HIV gag-pol sequences,wherein the database further provides a correlation between genotypesand phenotypes, wherein the correlation is indicative of efficacy of agiven drug regimen. A database of gag-pol sequences may be created andused as described in WO 01/79540. For example, such a database may beanalyzed in combination with gag, pol, protease, reverse transcriptaseor integrase sequence information and the results used in thedetermination of appropriate treatment strategies. Said databasecontaining a collection of genotypes, phenotypes and samples for whichthe combined genotype/phenotype are available, may be used to determinethe virtual phenotype (see supra). In addition, instead of interrogatingthe complete gag-pol sequences, particular codons correlating to achange in drug susceptibility of the virus may be interrogated in suchdatabase.

A primer may be chosen from SEQ ID N^(o) 1-10, 53 and 54. A particularset of primers is SEQ ID 1-4 and 53 and 54. Primers specific for thegag-pol region of the HIV genome such as the primers described hereinand their homologs are disclosed to perform the assay according to theinvention. The primer sequences listed herein may be labelled. Suitably,this label may be detected using fluorescence, luminescence orabsorbance. The primer for creating a deletion construct may contain aportion that does not anneal to the HIV sequence. That portion may beused to introduce a unique restriction site. Interestingly, primers maybe designed in which the unique restriction site is partially present inthe HIV sequence. The primers are chosen from those listed herein orhave at least 80% homology as determined by methods known by the personskilled in the art such BLAST or FASTA. Specifically, the homology is atleast 90%, more specifically, at least 95%. In addition, primers locatedin a region of 50 nucleotides (nt) upstream or downstream from thesequences given herein constitute part of the invention. Especially,said region is 20 nucleotides up or downstream from the position in theHIV genome of the primer sequences given herein. Alternatively, primerscomprising at least 8 consecutive bases present in either of the primersdescribed here constitute one embodiment of the invention.Interestingly, the primers comprise at least 12 consecutive basespresent in either of the primers described herein.

Examples

General Outline

An amplicon was generated from patient-derived plasma viral RNA byRT-PCR and nested PCR. This amplicon, further referred to as 5′LTR-Viffragment, contains the complete Gag and complete Pol (PR-RT-INT) codingregion (4588 bp). Sequence primers across the 5′ end of HIV-1 allow fornucleotide sequence determination and genotypic drug resistanceanalysis.

A delta[Gag-Pol] backbone (SEQ ID NO: 49) was made starting from anHIV-1 vector that contains eGFP in the Nef coding region. In vitrocloning (using BD In-Fusion™ Clontech Laboratories Inc.) between thePCR-generated amplicon and the delta[Gag-Pol] backbone resulted in afully replication-competent HIV-1 that was used in experiments toevaluate phenotypic drug resistance.

Further, an amplicon spanning the Gag cleavage sites p1/p7 and p1/p6,PR, RT, RNaseH and INT (3202 bp), referred to as Pol fragment, wasevaluated together with an amplicon containing the Gag and PR codingsequence (1980 bp), referred to as Gag-PR fragment, and an ampliconcontaining the complete RT, RNaseH and INT coding sequence (2898 bp),named RT-INT fragment.

For phenotypic evaluation delta[Pol] (SEQ ID NO: 52) delta[Gag-PR] (SEQID NO: 50) and delta[RT-INT] HIV-1 (SEQ ID NO: 51) backbones, alsocontaining eGFP (enhanced Green Fluorescent protein) in Nef, weredesigned respectively.

Protocol for Amplification of 5′LTR-VIF Fragment

Starting from freshly prepared patient-derived RNA, 5 μl was mixed with0.2 μM forward outer primer (5LTR_IF1=SEQ ID NO: 1) and 0.2 μM reverseouter primer (VIF_R2=SEQ ID NO: 2), 1× Superscript™ reaction buffer(containing 0.4 mM of each dNTP and 2.5 mM MgSO₄) and 0.5 μlSuperscript™ III HIFI enzyme mix in a total volume of 25 μl (Table 1).The reverse transcription reaction was performed @ 53° C. for 30 min,followed by an initial denaturation @ 94° C. for 2 min. This wasfollowed by 30 cycles of [denaturation @ 92° C. for 15 sec, annealing @55° C. for 30 sec and elongation @ 68° C. for 5 min]. The finalelongation step was 10 min @ 68° C. (Table 2).

Subsequently, 1 μl of outer PCR product was mixed with 0.304 μM forwardinner primer (5LTR_F2=SEQ ID NO: 3) and 0.304 μM reverse inner primer(VIF_R5=SEQ ID NO: 4), 1× Expand™ HIFI reaction buffer, 0.2 μl dNTP's(0.2 mM) and 0.3 μl Expand™ HIFI enzyme mix (=1.05 U) in a total volumeof 25 μl (Table 1).

The inner PCR reaction consists of an initial denaturation @ 94° C. for2 min, followed by 35 cycles of [denaturation @ 94° C. for 15 sec,annealing @ 61° C. for 30 sec and elongation @ 68° C. for 5 min]. Thefinal elongation step was 10 min @ 68° C. (Table 2).

All reaction mixtures and samples were kept on ice during preparation.The outer and inner primers used to generate this amplicon can be foundin Table 7.

Finally, 4 μl PCR product was mixed with 2 μl loading dye, loaded on a1% agarose gel and stained with ethidium bromide for visualization.

TABLE 1 Composition of the RT-outer PCR mix and inner PCR mix foramplification of the 5′LTR-VIF fragment. RT-outer PCR mix inner PCR mixvolume/ volume/ sample sample component (μl) component (μl) DEPC.water6.5 DEPC.water 20.24 2 x reaction buffer 12.5 10 x reaction buffer 2.55LTR_IF1 primer 0.25 5LTR_F2 primer (20 μM) 0.38 (20 μM) VIF_R2 primer(20 μM) 0.25 VIF_R5 primer (20 μM) 0.38 Superscript III HiFi 0.5 dNTP's(25 mM) 0.2 RNA 5 Expand HiFi (3.5 U/μl) 0.3 total volume (μl) 25OUT_sample 1 total volume (μl) 25

TABLE 2 Thermal cycling conditions for the outer and inner PCR foramplification of the 5′LTR-VIF fragment. step temperature (° C.) timecycles outer PCR 5′LTR-VIF fragment 1 53 30 min 2 94 2 min 3 92 15 s 304 55 30 s 5 68 5 min 6 68 10 min 7 4 hold inner PCR 5′LTR-VIF fragment 194 2 min 2 94 15 s 35 3 61 30 s 4 68 5 min 5 68 10 min 6 4 hold

Protocol for Amplification of Pol Fragment

Starting from freshly prepared patient-derived RNA, 5 μl was mixed with0.2 μM forward outer primer (5′OUT=SEQ ID NO: 53) and 0.2 μM reverseouter primer (VIF_R2=SEQ ID NO: 2), 1× Superscript™ reaction buffer(containing 0.4 mM of each dNTP and 2.5 mM MgSO₄) and 0.5 μlSuperscript™ III HIFI enzyme mix in a total volume of 25 μl (Table 12).The reverse transcription reaction was performed @ 53° C. for 30 min,followed by an initial denaturation @ 94° C. for 2 min. This wasfollowed by 30 cycles of [denaturation @ 92° C. for 15 sec, annealing @55° C. for 30 sec and elongation @ 68° C. for 3 min 30 sec]. The finalelongation step was 10 min @ 68° C. (Table 13).

Subsequently, 1 μl of outer PCR product was mixed with 0.304 μM forwardinner primer (5′IN=SEQ ID NO: 54) and 0.304 μM reverse inner primer(VIF_R5=SEQ ID NO: 4), 1× Expand™ HIFI reaction buffer, 0.2 μl dNTP's(0.2 mM) and 0.3 μl Expand™ HIFI enzyme mix (=1.05 U) in a total volumeof 25 μl (Table 12).

The inner PCR reaction consists of an initial denaturation @ 94° C. for2 min, followed by 35 cycles of [denaturation @ 94° C. for 15 sec,annealing @ 58° C. for 30 sec and elongation @ 68° C. for 3 min 30 sec].The final elongation step was 10 min @ 68° C. (Table 13).

All reaction mixtures and samples were kept on ice during preparation.The outer and inner primers used to generate this amplicon can be foundin Table 7.

Finally, 4 μl PCR product was mixed with 2 μl loading dye, loaded on a1% agarose gel and stained with ethidium bromide for visualization.

TABLE 12 Composition of the RT-outer PCR mix and inner PCR mix foramplification of the Pol fragment. RT-outer PCR mix inner PCR mixvolume/ volume/ sample sample component (μl) component (μl) DEPC.water6.5 DEPC.water 20.24 2 x reaction buffer 12.5 10 x reaction buffer 2.55LTR_IF1 primer 0.25 5LTR_F2 primer (20 μM) 0.38 (20 μM) VIF_R2 primer(20 μM) 0.25 VIF_R5 primer (20 μM) 0.38 Superscript III HiFi 0.5 dNTP's(25 mM) 0.2 RNA 5 Expand HiFi (3.5 U/μl) 0.3 total volume (μl) 25OUT_sample 1 total volume (μl) 25

TABLE 13 Thermal cycling conditions for the outer and inner PCR foramplification of the Pol fragment. step temperature (° C.) time cyclesouter PCR Pol fragment 1 53 30 min 2 94 2 min 3 92 15 s 30 4 55 30 s 568 3 min 30 sec 6 68 10 min 7 4 hold inner PCR Pol fragment 1 94 2 min 294 15 s 35 3 58 30 s 4 68 3 min 30 sec 5 68 10 min 6 4 hold

Protocol for Amplification of RT-INT Fragment

Starting from freshly prepared patient-derived RNA, 5 μl was mixed with0.2 μM forward outer primer (PR_F1=SEQ ID NO: 5) and 0.2 μM reverseouter primer (VIF_R3=SEQ ID NO: 6), 1× Superscript™ reaction buffer(containing 0.4 mM of each dNTP and 2.5 mM MgSO₄) and 0.5 μlSuperscript™ III HIFI enzyme mix in a total volume of 25 μl (Table 3).The reverse transcription reaction was performed @ 56° C. for 30 min,followed by an initial denaturation @ 94° C. for 2 min. This wasfollowed by 30 cycles of [denaturation @ 92° C. for 15 sec, annealing @62° C. for 30 sec and elongation @ 68° C. for 3 min 30 sec]. The finalelongation step was 10 min @ 68° C. (Table 4).

Subsequently, 1 μl of outer PCR product was mixed with 0.304 μM forwardinner primer (PR_F3=SEQ ID NO: 7) and 0.304 μM reverse inner primer(VIF_R5=SEQ ID NO: 4), 1× Expand™ HIFI reaction buffer, 0.2 μl dNTP's(0.2 mM) and 0.3 μl Expand™ HIFI enzyme mix (=1.05 U) in a total volumeof 25 μl (Table 3)

The inner PCR reaction consists of an initial denaturation @ 94° C. for2 min, followed by 35 cycles of [denaturation @ 94° C. for 15 sec,annealing @ 60° C. for 30 sec and elongation @ 68° C. for 3 min]. Thefinal elongation step was 10 min @ 68° C. (Table 4).

All reaction mixtures and samples were kept on ice during preparation.The outer and inner primers used to generate this amplicon can be foundin Table 7.

Finally, 4 μl PCR product was mixed with 2 μl loading dye, loaded on a1% agarose gel and stained with ethidium bromide for visualization.

TABLE 3 Composition of the RT-outer PCR mix and inner PCR mix foramplification of the RT-INT fragment. RT-outer PCR mix inner PCR mixvolume/ volume/sample component sample (μl) component (μl) DEPC.water6.5 DEPC.water 20.24 2 x reaction buffer 12.5 10 x reaction buffer 2.5PR_F1 (20 μM) 0.25 PR_F3 (20 μM) 0.38 VIF_R3 (20 μM) 0.25 VIF_R5 (20 μM)0.38 Superscript III HiFi 0.5 dNTP's (25 mM) 0.2 RNA 5 Expand HiFi (3.5U/ 0.3 μl) total volume (μl) 25 OUT_sample 1 total volume (μl) 25

TABLE 4 Thermal cycling conditions for the outer and inner PCR foramplification of the RT-INT fragment. step temperature (° C.) timecycles outer PCR RT-INT fragment 1 53 30 min 2 94 2 min 3 92 15 s 30 455 30 s 5 68 3 min 30 s 6 68 10 min 7 4 hold inner PCR RT-INT fragment 194 2 min 2 94 15 s 35 3 60 30 s 4 68 3 min 5 68 10 min 6 4 hold

Protocol for Amplification of GAG-PR Fragment

Starting from freshly prepared patient-derived RNA, 5 μl was mixed with0.2 μM forward outer primer (EF1=SEQ ID NO: 8) and 0.2 μM reverse outerprimer (Gaprout-R3=SEQ ID NO: 9), lx Superscript™ reaction buffer(containing 0.4 mM of each dNTP and 2.5 mM MgSO₄) and 0.5 μlSuperscript™ III HIFI enzyme mix in a total volume of 25 μl (Table 5).The reverse transcription reaction was performed @ 53° C. for 30 min,followed by an initial denaturation @ 94° C. for 2 min. This wasfollowed by 30 cycles of [denaturation @ 92° C. for 15 sec, annealing @55° C. for 30 sec and elongation @ 68° C. for 2 min 30 sec]. The finalelongation step was 10 min @ 68° C. (Table 6).

Subsequently, 1 μl of outer PCR product was mixed with 0.304 μM forwardinner primer (5LTR_IF1=SEQ ID NO:1) and 0.304 μM reverse inner primer(Gaprout-R1=SEQ ID NO: 10), 1× Expand™ HIFI reaction buffer, 0.2 μldNTP's (0.2 mM) and 0.3 μl Expand™ HIFI enzyme mix (=1.05 U) in a totalvolume of 25 μl (Table 5).

The inner PCR reaction consists of an initial denaturation @ 94° C. for2 min, followed by 35 cycles of [denaturation @ 94° C. for 15 sec,annealing @ 60° C. for 30 sec and elongation @ 72° C. for 2 min]. Thefinal elongation step was 10 min @ 72° C. (Table 6).

All reaction mixtures and samples were kept on ice during preparation.The outer and inner primers used to generate this amplicon can be foundin Table 7.

Finally, 4 μl PCR product was mixed with 2 μl loading dye, loaded on a1% agarose gel and stained with ethidium bromide for visualization.

TABLE 5 Composition of the RT-outer PCR mix and inner PCR mix foramplification of the GAG-PR fragment. RT-outer PCR mix inner PCR mixvolume/ volume/sample component sample (μl) component (μl) DEPC.water6.5 DEPC.water 20.24 2 x reaction buffer 12.5 10 x reaction buffer 2.5EF1 (20 μM) 0.25 5LTR_IF1 (20 μM) 0.38 Gaprout-R3 (20 μM) 0.25Gaprout-R1 (20 μM) 0.38 Superscript III HiFi 0.5 dNTP's (25 mM) 0.2 RNA5 Expand HiFi (3.5 U/ 0.3 μl) total volume (μl) 25 OUT_sample 1 totalvolume (μl) 25

TABLE 6 Thermal cycling conditions for the outer and inner PCR foramplification of the GAG-PR fragment. step temperature (° C.) timecycles outer PCR Gag-PR fragment 1 53 30 min 2 94 2 min 3 92 15 s 30 455 30 s 5 68 2 min 30 s 6 68 10 min 7 4 hold inner PCR Gag-PR fragment 194 2 min 2 94 15 s 35 3 60 30 s 4 72 2 min 5 72 10 min 6 4 hold

TABLE 7 Primer sequences of all amplification primers and their positionon the HXB2 reference. primer name primer sequence from 5′ to 3′position on HXB2 EF1 CAA GTA GTG TGT GCC CGT CTG T 550-571 5LTR_IF1TGG AAA ATC TCT AGC AGT GGC G 619-640 5LTR_F2TCT CTA GCA GTG GCG CCC GAA CA 626-648 PR_F1CCC TCA AAT CAC TCT TTG GCA ACG AC 2252-2277 PR_F3GCT CTA TTA GAT ACA GGA GCA GAT G 2316-2340 VIF_R2AGT GGG ATG TGT ACT TCT GAA C 5195-5216 VIF_R3CTC CTG TAT GCA GAC CCC AAT ATG 5243-5266 VIF_R5GGG ATG TGT ACT TCT GAA CTT 5193-5213 Gaprout-R3CCA TTG TTT AAC TTT TGG GCC ATC C 2597-2621 Gaprout-R1CCA TTC CTG GCT TTA ATT TTA CTG G 2574-2598 5′ OUTGCC CCT AGG AAA AAG GGC TGT TGG 2008-2031 5′ INCTA GGA AAA AGG GCT GTT GGA AAT G 2012-2036

SEQ ID NO 1: (5LTR_IF1) TGG AAA ATC TCT AGC AGT GGC GSEQ ID NO 2: (VIF_R2) AGT GGG ATG TGT ACT TCT GAA CSEQ ID NO 3: (5LTR_F2) TCT CTA GCA GTG GCG CCC GAA CASEQ ID NO 4: (VIF_R5) GGG ATG TGT ACT TCT GAA CTT SEQ ID NO 5: (PR_F1)CCC TCA AAT CAC TCT TTG GCA ACG AC SEQ ID NO 6: (VIF_R3)CTC CTG TAT GCA GAC CCC AAT ATG SEQ ID NO 7: (PR_F3)GCT CTA TTA GAT ACA GGA GCA GAT G SEQ ID NO 8: (EF1)CAA GTA GTG TGT GCC CGT CTG T SEQ ID NO 9: (Gaprout-R3)CCA TTG TTT AAC TTT TGG GCC ATC C SEQ ID NO 10: (Gaprout-R1)CCA TTC CTG GCT TTA ATT TTA CTG G SEQ ID NO 53 (5′OUT)GCC CCT AGG AAA AAG GGC TGT TGG SEQ ID NO 54 (5′IN)CTA GGA AAA AGG GCT GTT GGA AAT G

Sequencing Protocol for all Fragments Mentioned Before

Sequencing reactions were performed with the Big Dye Terminator CycleSequencing Kit v3.1 (Applied Biosystems). Each reaction mixture (11.5μl) contained: the amplicon (1 DNase RNase free water (3 μl), Big Dyeterminator mix (1 μl), primer (4 μl, 4 μM) and 1× dilution buffer (1.0 MTris HCL, 1.0 M MgCl₂ and H₂O) (Table 8). All primers used fornucleotide sequencing of the different fragments are listed in Table 10.

The PCR conditions were 25 cycles of [10 seconds at 96° C., 5 seconds at50° C. and 4 minutes at 60° C.], followed by a final hold at 4° C. andusing an ABI 9800 Fast Thermal Cycler (Applied Biosystems) (Table 9).

The purification of the sequencing reaction mixtures was performed usingthe DyeEX (Qiagen) purification kit according to the manufacturersprotocol. The sequencing was performed using an ABI3730 XL (AppliedBiosystems) and the generated sequences were aligned and analyzed usingSeqScape v2.5 software (Applied Biosystems).

TABLE 8 Composition of the sequencing reaction mixture. mix compositionfor sequencing component vol/sample (μl) DEPC.water 3 2.5x dilutionbuffer 2.5 Big Dye terminator mix 1 primer (4 μM) 4 template 1 totalvolume (μl) 11.5

TABLE 9 Thermal cycling conditions for the sequencing reaction. thermalcycling program step temp. time # cycles 1 96° C. 10 s 25 2 50° C.  5 s3 60° C.  4 min 4  4° C. hold

TABLE 10Primer sequences of all sequencing primers and their position on the HXB2reference. Primer name P Nucleotide sequence (5′→3′) Position on HXB2Forward Primers SEQ ID NO 11 5′LTR_F_631 AGCAGTGGCGCCCGAACAG 631-649SEQ ID NO 12 F0 gag TTTGACTAGCGGGAGGCTAGAAG 761-782SEQ ID NO 13 GAG_F_1070 TAAAAGACACCAAGGAAGC 1070-1088SEQ ID NO 14 F10 gag AAGACACCAAGGAAGC 1073-1088 SEQ ID NO 15 F3 gagCATAGCAGGAACTACTAGTA 1494-1513 SEQ ID NO 16 GAG_F_1602TAAAATAGTAAGAATGTATAGCCC 1602-1625 SEQ ID NO 17 F5 gagATGACAGCATGTCAGGGAGT 1828-1847 SEQ ID NO 18 F1 GAGAGCTTCAGGTTTGGGG2170-2188 SEQ ID NO 19 F5 CACTCTTTGGCAACGACCC 2261-2279SEQ ID NO 20 PR_F2376 TGGAAACCAAAAATGATAGG 2376-2395 SEQ ID NO 21 F2AATTGGGCCTGAAAATCC 2696-2713 SEQ ID NO 22 F3 CCTCCATTCCTTTGGATGGG3222-3241 SEQ ID NO 23 RT_F_3681 GAAAGCATAGTAATATGGG 3681-3699SEQ ID NO 24 IN_F_4074 CAACCAGATAAAAGTGAATCAG 4074-4095SEQ ID NO 25 IN_F_4540 TAGCAGGAAGATGGCCAGT 4540-4558SEQ ID NO 26 Inseq3F GTAGACATAATAGCAACAGAC 4830-4850 SEQ ID NO 55 F7GTACTGGATGTGGGTGATGC 2871-2890 SEQ ID NO 56 F8 GTGGGAAAATTGAATTGGG3330-3348 SEQ ID NO 57 F3771 GCCACCTGGATTCCTGAGTG 3771-3790Reverse primers SEQ ID NO 27 R8 gag TCTTGTGGGGTGGCTCCTTC 1337-1318SEQ ID NO 28 GAG_R_1316 TCTTGTGGGGTGGCTCCTTCTG 1337-1316SEQ ID NO 29 R3 gag TCTACATAGTCTCTAAAGGG 1682-1663SEQ ID NO 30 GAG_R_1825 ACTCCCTGACATGCTGTCATCAT 1847-1825SEQ ID NO 31 R7 gag GTGGGGCTGTTGGCTCTGGT 2164-2145SEQ ID NO 32 PR_R_2382 ATTCCCCCTATCATTTTTGG 2401-2382 SEQ ID NO 33 R8GATAAAACCTCCAATTCC 2414-2397 SEQ ID NO 34 R3 CTTCCCAGAAGTCTTGAGTTC2817-2797 SEQ ID NO 35 R6 GGAATATTGCTGGTGATCC 3030-3012SEQ ID NO 36 RT_R_3304 TGTATGTCATTGACAGTCC 3322-3304 SEQ ID NO 37 R5GGGTCATAATACACTCCATG 3511-3492 SEQ ID NO 38 R1 CTCCCACTCAGGAATCC3794-3778 SEQ ID NO 39 RT_R_3964 CAGTCTTCTGATTTGTTG 3981-3964SEQ ID NO 40 RT_R_4150 CTTTGTGTGCTGGTACCCATG 4170-4150SEQ ID NO 41 RT_R_4380a GGACTACAGTCTACTTGTCCAATG 4402-4380SEQ ID NO 42 Inseq2R CTGCCATTTGTACTGCTGTC 4767-4748SEQ ID NO 43 IN_R_5042 ATCACCTGCCATCTGITTICCA 5063-5042SEQ ID NO 44 VIF_R_5193 ATGTGTACTTCTGAACTT 5210-5193SEQ ID NO 58 IN_R_4348 CTCCTTTTAGCTGACATTTATCAC 4371-4348

Creation of the HXB2D_eGFP_delta[GAG-POL] Backbone (SEQ ID NO: 49)

This backbone contains all genetic elements of HIV-1, except thecomplete GAG and POL region. Recombination between this GAG-POL deletionbackbone and the 5′LTR-VIF amplicon resulted in a fully functional HIV-1viral vector, which was used in transfection/infection experiments.

First, pUC18 was digested with PstI and EcoRI restriction enzymes.Subsequently, a 35 bp synthetic linker containing the HpaI, SpeI, andSalI restriction sites was ligated into the PstI/EcoRI-linearized pUC18plasmid, creating pUC18-LINK. Next, HXB2D_eGFP (original fullyreplication competent HIV-1 vector, containing eGFP in Nef) was digestedwith HpaI and SalI (termed vector C), cutting out the 5′ part of theHIV-1 genome (5′LTR, GAG, POL, VIF) (from nucleotide 15223 to 5786compared to the HXB2 reference), termed fragment A. Fragment A was thencloned into the HpaI/SalI-digested pUC18-LINK plasmid. PCR primers thatare complementary to the 5′ and 3′ ends of the 5′LTR-Vif amplicon weredesigned and used in an ‘inverse PCR’ (iPCR) reaction to ‘re-create’ thenucleotide sequence that was removed in excess during HpaI/SalIdigestion (i.e. sequence between primer binding site and restrictionsite). These inverse PCR primers were extended with the nucleotidesequences of two restriction sites (i.e. PacI and SnaBI) forlinearization of the backbone afterwards. Finally, HpaI/SalI digestionwas performed on the iPCR product and the excised HpaI/SalI fragment(fragment B) was cloned back into the HpaI/SalI digested originalHXB2D-eGFP vector (vector C) (see FIG. 1, 2, 3).

Creation of the HXB2D_eGFP_delta[POL] Backbone (SEQ ID NO: 52)

This backbone contains all genetic elements of HIV-1, except thecomplete Pol region. Recombination between this Pol deletion backboneand the Pol amplicon resulted in a fully functional HIV-1 viral vector,which was used in transfection/infection experiments.

First, pUC18 was digested with PstI and EcoRI restriction enzymes.Subsequently, a 35 bp synthetic linker containing the HpaI, SpeI, andSalI restriction sites was ligated into the PstI/EcoRI-linearized pUC18plasmid, creating pUC18-LINK. Next, HXB2D_eGFP (original fullyreplication competent HIV-1 vector, containing eGFP in Nef) was digestedwith HpaI and SalI (termed vector C), cutting out the 5′ part of theHIV-1 genome (5′LTR, GAG, POL, VIF) (from nucleotide 15223 to 5786compared to the HXB2 reference), termed fragment A. Fragment A was thencloned into the HpaI/SalI-digested pUC18-LINK plasmid. PCR primers thatare complementary to the 5′ and 3′ ends of the Pol amplicon weredesigned and used in an ‘inverse PCR’ (iPCR) reaction to ‘re-create’ thenucleotide sequence that was removed in excess during HpaI/SalIdigestion (i.e. sequence between primer binding site and restrictionsite). These inverse PCR primers were extended with the nucleotidesequences of two restriction sites (i.e. Pad and SnaBI) forlinearization of the backbone afterwards. Finally, HpaI/SalI digestionwas performed on the iPCR product and the excised HpaI/SalI fragment(fragment P) was cloned back into the HpaI/SalI digested originalHXB2D-eGFP vector (vector C) (see FIGS. 12 and 13).

Creation of the HXB2D_eGFP_delta[RT-INT] Backbone (SEQ ID NO: 51)

This backbone contains all genetic elements of HIV-1, except thecomplete RT and INT region. Recombination between this RT-INT deletionbackbone and the RT-INT amplicon resulted in a fully functional HIV-1viral vector, which was used in transfection/infection experiments.

First, pUC18 was digested with PstI and EcoRI restriction enzymes.Subsequently, a 35 bp synthetic linker containing the HpaI, SpeI, andSalI restriction sites was ligated into the PstI/EcoRI-linearized pUC18plasmid, creating pUC18-LINK. Next, HXB2D_eGFP (original fullyreplication competent HIV-1 vector, containing eGFP in Nef) was digestedwith SpeI and SalI (termed vector Z), cutting out the majority of POLand VIF of the HIV-1 genome (from nucleotide 1507 to 5786 compared tothe HXB2 reference), termed fragment X. Fragment X was then cloned intothe SpeI/SalI-digested pUC18-LINK plasmid. PCR primers that arecomplementary to the 5′ and 3′ ends of the RT-INT amplicon were designedand used in an ‘inverse PCR’ (iPCR) reaction to ‘re-create’ thenucleotide sequence that was removed in excess during SpeI/SalIdigestion (i.e. sequence between primer binding site and restrictionsite).

Finally, SpeI/SalI digestion was done on the iPCR product and theexcised SpeI/SalI fragment (fragment Y) was cloned back into theSpeI/SalI digested original HXB2D-eGFP vector (vector Z) (see FIG. 4, 5,6).

Creation of the HXB2D_eGFP_delta[GAG-PR] Backbone (SEQ ID NO: 50)

This backbone contains all genetic elements of HIV-1, except thecomplete GAG and PR region. Recombination between this GAG-PR deletionbackbone and the GAG-PR amplicon resulted in a fully functional HIV-1viral vector, which was used in transfection/infection experiments.

First, pUC18 was digested with PstI and EcoRI restriction enzymes.Subsequently, a 35 bp synthetic linker containing the HpaI, SpeI, andSalI restriction sites was ligated into the PstI/EcoRI-linearized pUC18plasmid, creating pUC18-LINK. Next, HXB2D_eGFP (original fullyreplication competent HIV-1 vector, containing eGFP in Nef) was digestedwith HpaI and SalI (termed vector C), cutting out the 5′ part of theHIV-1 genome (5′LTR, GAG, POL, VIF) (from nucleotide 15223 to 5786compared to the HXB2 reference), termed fragment A. Fragment A was thencloned into the HpaI/SalI-digested pUC18-LINK plasmid. PCR primers thatare complementary to the 5′ and 3′ ends of the GAG-PR amplicon weredesigned and used in an ‘inverse PCR’ (iPCR) reaction to ‘re-create’ thenucleotide sequence that was removed in excess during HpaI/SalIdigestion (i.e. sequence between primer binding site and restrictionsite). Finally, HpaI/SalI digestion was done on the iPCR product and theexcised HpaI/SalI fragment (fragment ALPHA) was cloned back into theHpaI/SalI digested original HXB2D-eGFP vector (vector C) (see FIG. 7, 8,9).

Phenotypic Assay Approach

After linearization of the Gag-Pol, Gag-PR and RT-INT HXB2D_eGFP_deltabackbone described before, the respective purified amplicon was clonedin the appropriate backbone using the In-Fusion™ technology (Clontech,Mountain view, California) and subsequently transformed into MAXEfficiency® Stbl2™ cells (Invitrogen, Merelbeke, Belgium). After DNApreparation from either clones or the complete plate, full-lengthrecombinant HIV genomes were transfected to MT4 cells. At full CPE,recombinant virus stocks were harvested, titrated and subjected to anantiviral experiment.

The 3 phenotyping assays (GAG-POL, GAG-PR and RT-INT) are described inthe following section. The layout of the experiments is shown in FIG.10.

The three backbones, HXB2D_eGFP_delta [GAG-POL] (SEQ ID NO: 49),HXB2D_eGFP_delta [GAG-PR] (SEQ ID NO: 50) and HXB2D_eGFP_delta [RT-INT](SEQ ID NO: 51) were linearized by digestion with SnaBI and PacI. Afterpurification, for each backbone, 100 ng linearized vector was recombinedwith three different PCR amplicons (3× 5′LTR-VIF, 3× GAG-PR or 3× RT-INTamplicons) in vector/insert molar ratio of 1/10 using In-Fusionreagents. Thereafter, In-Fusion mixes were transformed to MAX EfficiencyStbl2 cells and incubated for 24 h at 30° C. The day after, colonieswere screened for the presence of the full recombinant plasmid by aduplex colony PCR using the primers shown in Table 11 (SEQ ID NO's:45-48).

As an example, full recombinants generate two fragments (493 bp and 217bp), while recircularized vectors containing no inserts, generate onlyone fragment (300 bp for

delta_[GAG-POL], 200 bp for delta_[GAG-PR] and 500 bp fordelta_[RT-INT]).

In general full-length HIV recombinants were obtained for all backbonesand for all amplicons tested.

For the GAG-POL assay, two full recombinants were obtained for sample 1and 2, and three recombinants for sample 3.

For the GAG-PR backbone, two recombinants were generated for sample 1,one recombinant for sample 2 and eight recombinants for sample 3.

For the RT-INT backbone, five full recombinants for sample 1, elevenrecombinants for sample 2 and two for sample 3 were generated.

All recombinant clones (with a maximum of five per sample) were grownovernight in LB-ampicillin at 30° C. to prepare DNA from (27 in total).Miniprep DNA was prepared using the Qiaprep Spin miniprep (Qiagen) andchecked by HindIII restriction digest. By comparison of the HindIIIdigestion pattern of the clones with that of the deletion backbones andthat of the full-length parental HXB2D_eGFP vector, all 27 clonescontained full-length HIV genomes. All 27 clones were transfected to MT4cells using the Amaxa nucleofection technique and evaluated for theircythopathic effect (CPE). In total, 18 clones reached full CPE(cyto-pathogenic effect) during the time of evaluation (11 days) andwere used for further infection experiments: 16 clones generated fullCPE after 4 days, 1 clone after 5 days and 1 clone after 11 days. Theother 9 clones did not show substantial infection after 11 days and werestopped for further analysis. The 18 harvested RVS (recombinant virusstock) were titrated and subjected to an antiviral experiment (AVE) at astandardized MOT (multiplicity of infection) using FDA-approved proteaseand RT inhibitors, and experimental maturation (PA-457) and integrase(GS-9137, L870,810 and L731,988) inhibitors. After 3 days of infection,GFP (green fluorescent protein) infection signals were quantified anddose-response curves were calculated. Only 1 out of 18 samples did notgenerate significant GFP expression above background, all other 17 RVSwere successfully phenotyped for all drugs tested. As an example, FIG.11 shows the dose-response curves 1 GAG-POL RVS for all drugs tested.

TABLE 11Primer sequences of the primers used for the colony PCR and their position onthe HXB2 reference. primer name primer sequence from 5′ to 3′Position on HXB2 SEQ ID NO 45: HXB2_5LTR_F_422CTG CAT ATA AGC AGC TGC TTT TTG 422-445 SEQ ID NO 46: GAG_R_895TCT AGC TCC CTG CTT GCC CA 895-914 SEQ ID NO 47: IN_F_5052ATG GCA GGT GAT GAT TGT GTG G 5052-5073 SEQ ID NO 48: HXB2_VIF_R_5247TTC TCC TGT ATG CAG ACC CCA A 5247-5268

What is claimed:
 1. An in vitro method according to claim 1 fordesigning a drug regime for an HIV-infected patient by determining thephenotypic susceptibility of HIV to at least one drug, comprising: i)using at least one sample comprising HIV RNA from a patient, wherein thesample comprises the complete HIV reverse transcriptase-integrase codingsequence; ii) reverse-transcribing and amplifying the HIV RNA withprimers specific for the complete HIV reverse transcriptase-integrasecoding sequence to obtain at least one amplicon comprising the completeHIV reverse transcriptase-integrase coding sequence, wherein at leastone primer is selected from SEQ ID NO: 4-7; iii) generating a plasmidcomprising a reference HIV sequence with a deletion of the complete HIVreverse transcriptase-integrase coding sequence; iv) preparing at leastone recombinant virus by recombination or ligation between at least oneamplicon obtained in step ii) and the plasmid comprising the referenceHIV sequence with a deletion of the complete HIV reversetranscriptase-integrase coding sequence obtained in step iii), and v)monitoring at least one recombinant virus in the presence of at leastone drug to determine the phenotypic susceptibility of HIV to at leastone drug, wherein said susceptibility is determined by thecytopathogenicity of said recombinant virus to cells or by determiningthe replicative capacity of said recombinant virus in the presence of atleast one drug.
 2. The method of claim 1 wherein said plasmid isHXB2D_eGFP_delta[RT-INT] (SEQ ID NO:51).
 3. An in vitro method accordingto claim 1 for designing a drug regime for an HIV-infected patient bydetermining the phenotypic susceptibility of HIV to at least one drug,comprising: i) using at least one sample comprising HIV DNA, wherein thesample comprises the complete HIV reverse transcriptase-integrase codingsequence; ii) amplifying the HIV DNA with primers specific for thecomplete HIV reverse transcriptase-integrase coding sequence to obtainat least one amplicon comprising the complete HIV reversetranscriptase-integrase coding sequence, wherein at least one primer isselected from SEQ ID NO: 4-7; iii) generating a plasmid comprising areference HIV sequence with a deletion of the complete HIV reversetranscriptase-integrase coding sequence; iv) preparing at least onerecombinant virus by recombination or ligation between at least oneamplicon obtained in step ii) and the plasmid comprising the referenceHIV sequence with a deletion of the complete HIV reversetranscriptase-integrase coding sequence obtained in step iii), and v)monitoring at least one recombinant virus in the presence of at leastone drug to determine the phenotypic susceptibility of HIV to at leastone drug, wherein said susceptibility is determined by thecytopathogenicity of said recombinant virus to cells or by determiningthe replicative capacity of said recombinant virus in the presence of atleast one drug.
 4. The method of claim 3 wherein said plasmid isHXB2D_eGFP_delta[RT-INT] (SEQ ID NO:51).